Application Of Tree Legume Rhizobia For Reforestation Program In Thailand Ii Biodiversity Of Tree Legume Rhizobia In Thailand

N. Teaumroong1, M. Manassila1, S. Kotepong2, A. Nuntagij2, N. Boonkerd1

1 School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima, Thailand

2Soil Microbiology Research Group, Division of Soil Science, Department of Agriculture, Thailand

Rhizobial strains from tree legumes were isolated from five varieties of native tree leguminous plants in all parts of Thailand, altogether with selected the highest N2-fixing efficiency strains. The 17 strains were isolated from Acacia auriculaformis, 52 strains from A. mangium, 16 strains from Millettia leucantha Kurz, 21 strains from Pterocarpus indicus Willd, and 42 strains from Xylia kerii Taub. Determination of growth rate and acid-base production on YM agar demonstrated only six strains were fast grower (two strains from A. auriculaformis, and four strains from A. mangium) and other strains were slow grower (base production). Indole Acetic Acid (IAA) production showed that nine strains could produced IAA (four strains from A. auriculaformis, three strains from A. mangium and two strains from X kerii). Nodulation with soybean, mungbean and cowpea were observed. Soybean was nodulated by two strains of X. kerii and A. auriculaformis rhizobia. Furthermore, mungbean and cowpea could be nodulated by 38 and 48 rhizobial strains, respectively. DNA from selected strains were amplified by BoxAIR primer. The strains which had differences in DNA fingerprint patterns were selected prior to determine the relationship by 16S rRNA and NodA-PCR primer. The size of PCR product obtain from 16S rRNA and NodA-PCA were 1500 bp and 700-900 bp, respectively. Determination between selected strains and reference strains by PCR-RFLP of Nod A and sequence of 16S rRNA gene found that some of the tree legume rhizobia were closely related to Bradyrhizobium elkanii strain USDA 94 and different from other reference rhizobial strains.


P. van Berkum1, B.D. Eardly2

'Soybean and Alfalfa Research Laboratory, USDA, ARS, Beltsville, MD 20705, USA 2Penn State Berks-Lehigh Valley, Berks Campus, PO Box 7009, Reading, PA 19610, USA

1. Introduction

The central focus of this study initially was to confirm the phylogenetic placement of bradyrhizobial isolates of Aeschynomene indica since they are more closely related to B. japonicum and other non-bradyrhizobial members of subgroup 2b than they are to B. elkanii. From our analysis, which included type strains representing the named genera in the alpha subdivision subgroup 2b, we concluded that some isolates were closely related to Bl. denitrificans. Therefore, we examined the possibility that the type strain of Bl. denitrificans would form a symbiotic relationship with A. indica.

2. Procedure

The strains used were Blastobacter denitrificans type strain IF AM 1005 (LMG 8443) kindly provided by the Belgian Culture Collection of Microorganisms, BTAil and USDA 4424 (van Berkum et al. 1995). Seeds of A. indica were surface sterilized and the plants were grown in a greenhouse without supplemental lighting for 34 days during July/August. Each of the cultures were tested for symbiosis. Each treatment was prepared in five replications and five jars without inoculated bacteria served as controls. Determination of nitrogenase activities was as described by van Berkum et al. 1995. The plant tops were dried at 60°C for two days to determine dry matter and total nitrogen contents. Nodules were used to isolate bacterial occupants in culture as described (van Berkum et al. 1995). Nodulation of A. indica by Bl. denitrificans in growth pouches was investigated in triplicate on two separate occasions as described by van Berkum et al. 1995.

3. Results and Discussion

All three cultures nodulated A. indica while uninoculated control plants formed no nodules. Nitrogen fixation in the inoculated plants was evident by their increased growth and by the presence of acetylene reduction activity in their roots. The symbiotic response of Bl. denitrificans was similar to that of USDA 4424 and was superior to that of BTAil. Isolates from nodules obtained from the Bl. denitrificans and the inoculated culture had identical ITS region sequences confirming that Bl. denitrificans formed the symbiosis. From this result the natural tendency might be to view the bradyrhizobia of A. indica as Blastobacter species or to consider changing the genus Blastobacter to Bradyrhizobium. However, it is important to examine both the taxonomic status of Blastobacter and the bradyrhizobia that include the phototrophs first.

4. References van Berkum P et al. (1995) Appl. Environ. Microbiol. 61, 623-629


F. Zakhia1, A. Bekki2, H. Jeder3, O. Domergue1, M. Gillis4, J. Cleyet-Marel1, B. Dreyfus1, P. de Lajudie1

^STM., IRD/CIRAD/INRA/AGRO-M, Montpellier, France 2Univ. Oran, Algeria 3IRA, Gabes, Tunisia 4Univ. Gent, Belgium

Demographic pressure and socio-economic changes in countries around the Mediterranean sea have major consequences on the ecosystems. Plant resources are decreasing due to overgrazing, soil erosion, aridity, salinity and desertification. To restore degraded lands and to face increased forage and agronomic needs, leguminous plants are good candidates: they are good colonizers, they establish nitrogen-fixing symbioses with rhizobial bacteria, they are resistant to aridity and salinity, and many produce human and animal food. We investigated nodulation of spontaneous legumes in North Africa (Tunisia and Algeria), belonging to several genera such as Acacia, Acmispon, Anthyttis, Argyrolobium, Astragalus, Callicotom, Colutea, Ebenus, Genista, Hedysarum, Hippocrepis, Lathyrus, Lotus, Medicago, Onobrychis, Ononis, Prosopis, Retama, Trigonella and Vicia. We isolated and characterized ca. 100 rhizobial bacteria from nodules of these plants sampled in different ecological regions. Diversity was estimated using SDS-PAGE of total cellular proteins and PCR-RFLP of rRNA operon. Some rhizobial isolates were identified as known Sinorhizobium and Mesorhizobium species, but others, from saline and/or arid zones, constituted potential new groups.

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